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Macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for alphavbeta3 and alphavbeta5 integrins, and protein kinase C regulates alphavbeta5 binding and cytoskeletal linkage.

Finnemann SC, Rodriguez-Boulan E - J. Exp. Med. (1999)

Bottom Line: In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors.Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments.These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Margaret M. Dyson Vision Institute, New York, New York 10021, USA. sfinne@mail.med.cornell.edu

ABSTRACT
Noninflammatory monocyte macrophages use alphavbeta3 integrin to selectively bind apoptotic cells, initiating their phagocytic removal. In a related process, the retinal pigment epithelium (RPE) employs alphavbeta5 integrin to recognize spent photoreceptor outer segment particles (OS). Here, we show that apoptotic cells and OS compete for binding to these receptors, indicating that OS and apoptotic cells expose surface signals recognizable by alphavbeta3 and alphavbeta5. Particle binding to alphavbeta5 required protein kinase C (PKC) activation. In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors. In macrophages, it was dormant but became activated upon PKC stimulation. PKC-activated alphavbeta5-mediated binding in macrophages differed from constitutive binding to the same integrin receptor in RPE cells in that the former followed much faster kinetics, similar to particle binding mediated by alphavbeta3. Activation of alphavbeta5 for particle binding correlated with its recruitment into a detergent-insoluble fraction, a process sensitive to pharmacological modulation of PKC in both types of phagocytes. Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments. These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

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Apoptotic cells and OS compete for binding to αvβ3 of macrophages and for binding to αvβ5 of RPE cells. (a) Time course of combined OS binding plus internalization by rat bone marrow–derived macrophages (x) and NRK-F49 fibroblasts (•) compared with RPE-J cell combined binding plus internalization of OS (▾) and apoptotic cells (▿). J774 binding and internalization were indistinguishable from those of rat primary macrophages. (b) Integrin dependence and RGD peptide inhibition of binding of OS (black bars) and apoptotic cells (white bars) by RPE-J cells and by J774 macrophages. Mean OS binding indices in the absence of apoptotic cells were 6.3 ± 0.9 for RPE-J and 3.8 ± 0.4 for J774, after 2 h or 30 min of particle challenge, respectively. At these time points, particle phagocytosis accounted for <20% of total particle count. Mean apoptotic cell binding indices in the absence of OS were 5.5 ± 0.4 for RPE-J and 3.7 ± 0.5 for J774, at the same time points. (c) Quantitative competition of OS with apoptotic cells for phagocyte binding. RPE-J and J774 macrophages were challenged with different ratios of OS and apoptotic cells, for 2 h or 30 min, respectively. Particles were labeled with different fluorophores (FITC and Texas red) and quantified by fluorescence scanning. 100% represents binding of each particle in the absence of the other; for binding indices, see b. OS, black bars; apoptotic cells, white bars. Results represent means ± SEM (n = 5). When challenged with equal numbers of OS and apoptotic cells, both phagocytes seem to prefer apoptotic cells. However, this may be attributed to the larger size of apoptotic cells compared with OS (see Fig. 1).
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Figure 2: Apoptotic cells and OS compete for binding to αvβ3 of macrophages and for binding to αvβ5 of RPE cells. (a) Time course of combined OS binding plus internalization by rat bone marrow–derived macrophages (x) and NRK-F49 fibroblasts (•) compared with RPE-J cell combined binding plus internalization of OS (▾) and apoptotic cells (▿). J774 binding and internalization were indistinguishable from those of rat primary macrophages. (b) Integrin dependence and RGD peptide inhibition of binding of OS (black bars) and apoptotic cells (white bars) by RPE-J cells and by J774 macrophages. Mean OS binding indices in the absence of apoptotic cells were 6.3 ± 0.9 for RPE-J and 3.8 ± 0.4 for J774, after 2 h or 30 min of particle challenge, respectively. At these time points, particle phagocytosis accounted for <20% of total particle count. Mean apoptotic cell binding indices in the absence of OS were 5.5 ± 0.4 for RPE-J and 3.7 ± 0.5 for J774, at the same time points. (c) Quantitative competition of OS with apoptotic cells for phagocyte binding. RPE-J and J774 macrophages were challenged with different ratios of OS and apoptotic cells, for 2 h or 30 min, respectively. Particles were labeled with different fluorophores (FITC and Texas red) and quantified by fluorescence scanning. 100% represents binding of each particle in the absence of the other; for binding indices, see b. OS, black bars; apoptotic cells, white bars. Results represent means ± SEM (n = 5). When challenged with equal numbers of OS and apoptotic cells, both phagocytes seem to prefer apoptotic cells. However, this may be attributed to the larger size of apoptotic cells compared with OS (see Fig. 1).

Mentions: Particle binding and phagocytosis proceeded with kinetics that were characteristic for each cell type. Macrophages bound and internalized OS with the same rapid and linear kinetics with which they take up apoptotic cells (Fig. 2 a, and data not shown). The early phase of RPE clearance of OS is characterized by a lag phase following particle challenge after which OS binding occurs rapidly 26. RPE cells bound apoptotic cells as slowly as OS (Fig. 2 a). After 2 h of phagocytic challenge, the sum of bound plus internalized OS was similar in macrophages and RPE cells (Fig. 2 a). However, at this time 78 ± 9% of these OS in macrophages had been internalized, compared with only 17 ± 4% internalized OS in RPE cells. Phagocytosis of apoptotic cells bound by RPE cells followed the same slow time course as previously established for OS: we removed unbound apoptotic cells after 2 h and followed the fate of bound cells. Over the next 3 h, 85% of bound apoptotic cells were internalized by RPE cells (26; and data not shown).


Macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for alphavbeta3 and alphavbeta5 integrins, and protein kinase C regulates alphavbeta5 binding and cytoskeletal linkage.

Finnemann SC, Rodriguez-Boulan E - J. Exp. Med. (1999)

Apoptotic cells and OS compete for binding to αvβ3 of macrophages and for binding to αvβ5 of RPE cells. (a) Time course of combined OS binding plus internalization by rat bone marrow–derived macrophages (x) and NRK-F49 fibroblasts (•) compared with RPE-J cell combined binding plus internalization of OS (▾) and apoptotic cells (▿). J774 binding and internalization were indistinguishable from those of rat primary macrophages. (b) Integrin dependence and RGD peptide inhibition of binding of OS (black bars) and apoptotic cells (white bars) by RPE-J cells and by J774 macrophages. Mean OS binding indices in the absence of apoptotic cells were 6.3 ± 0.9 for RPE-J and 3.8 ± 0.4 for J774, after 2 h or 30 min of particle challenge, respectively. At these time points, particle phagocytosis accounted for <20% of total particle count. Mean apoptotic cell binding indices in the absence of OS were 5.5 ± 0.4 for RPE-J and 3.7 ± 0.5 for J774, at the same time points. (c) Quantitative competition of OS with apoptotic cells for phagocyte binding. RPE-J and J774 macrophages were challenged with different ratios of OS and apoptotic cells, for 2 h or 30 min, respectively. Particles were labeled with different fluorophores (FITC and Texas red) and quantified by fluorescence scanning. 100% represents binding of each particle in the absence of the other; for binding indices, see b. OS, black bars; apoptotic cells, white bars. Results represent means ± SEM (n = 5). When challenged with equal numbers of OS and apoptotic cells, both phagocytes seem to prefer apoptotic cells. However, this may be attributed to the larger size of apoptotic cells compared with OS (see Fig. 1).
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Figure 2: Apoptotic cells and OS compete for binding to αvβ3 of macrophages and for binding to αvβ5 of RPE cells. (a) Time course of combined OS binding plus internalization by rat bone marrow–derived macrophages (x) and NRK-F49 fibroblasts (•) compared with RPE-J cell combined binding plus internalization of OS (▾) and apoptotic cells (▿). J774 binding and internalization were indistinguishable from those of rat primary macrophages. (b) Integrin dependence and RGD peptide inhibition of binding of OS (black bars) and apoptotic cells (white bars) by RPE-J cells and by J774 macrophages. Mean OS binding indices in the absence of apoptotic cells were 6.3 ± 0.9 for RPE-J and 3.8 ± 0.4 for J774, after 2 h or 30 min of particle challenge, respectively. At these time points, particle phagocytosis accounted for <20% of total particle count. Mean apoptotic cell binding indices in the absence of OS were 5.5 ± 0.4 for RPE-J and 3.7 ± 0.5 for J774, at the same time points. (c) Quantitative competition of OS with apoptotic cells for phagocyte binding. RPE-J and J774 macrophages were challenged with different ratios of OS and apoptotic cells, for 2 h or 30 min, respectively. Particles were labeled with different fluorophores (FITC and Texas red) and quantified by fluorescence scanning. 100% represents binding of each particle in the absence of the other; for binding indices, see b. OS, black bars; apoptotic cells, white bars. Results represent means ± SEM (n = 5). When challenged with equal numbers of OS and apoptotic cells, both phagocytes seem to prefer apoptotic cells. However, this may be attributed to the larger size of apoptotic cells compared with OS (see Fig. 1).
Mentions: Particle binding and phagocytosis proceeded with kinetics that were characteristic for each cell type. Macrophages bound and internalized OS with the same rapid and linear kinetics with which they take up apoptotic cells (Fig. 2 a, and data not shown). The early phase of RPE clearance of OS is characterized by a lag phase following particle challenge after which OS binding occurs rapidly 26. RPE cells bound apoptotic cells as slowly as OS (Fig. 2 a). After 2 h of phagocytic challenge, the sum of bound plus internalized OS was similar in macrophages and RPE cells (Fig. 2 a). However, at this time 78 ± 9% of these OS in macrophages had been internalized, compared with only 17 ± 4% internalized OS in RPE cells. Phagocytosis of apoptotic cells bound by RPE cells followed the same slow time course as previously established for OS: we removed unbound apoptotic cells after 2 h and followed the fate of bound cells. Over the next 3 h, 85% of bound apoptotic cells were internalized by RPE cells (26; and data not shown).

Bottom Line: In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors.Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments.These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Margaret M. Dyson Vision Institute, New York, New York 10021, USA. sfinne@mail.med.cornell.edu

ABSTRACT
Noninflammatory monocyte macrophages use alphavbeta3 integrin to selectively bind apoptotic cells, initiating their phagocytic removal. In a related process, the retinal pigment epithelium (RPE) employs alphavbeta5 integrin to recognize spent photoreceptor outer segment particles (OS). Here, we show that apoptotic cells and OS compete for binding to these receptors, indicating that OS and apoptotic cells expose surface signals recognizable by alphavbeta3 and alphavbeta5. Particle binding to alphavbeta5 required protein kinase C (PKC) activation. In RPE, alphavbeta5 binding was maximally activated even before any phagocytic challenge and was reduced by PKC inhibitors. In macrophages, it was dormant but became activated upon PKC stimulation. PKC-activated alphavbeta5-mediated binding in macrophages differed from constitutive binding to the same integrin receptor in RPE cells in that the former followed much faster kinetics, similar to particle binding mediated by alphavbeta3. Activation of alphavbeta5 for particle binding correlated with its recruitment into a detergent-insoluble fraction, a process sensitive to pharmacological modulation of PKC in both types of phagocytes. Furthermore, alphavbeta5 but not alphavbeta3 particle binding required actin microfilaments. These data constitute the first evidence that noninflammatory phagocytes actively regulate the earliest phase of phagocytic clearance, particle binding, by controlling receptor activity.

Show MeSH
Related in: MedlinePlus